Botulism is caused by Clostridium botulinum neurotoxin (BoNT), a CDC Category A biodefense threat agent for which no antidote exists. We have developed two distinct "designer E3-ligases" that target either the intracellular proteases of botulinum neurotoxin serotype A or B (BoNT/A, BoNT/B) for accelerated ubiquitin- mediated degradation. Our agents consist of the F-box domain of TrCP fused to a came lid VHH domain with binding specificity for the BoNT protease. These polypeptide agents, with a size less than 30 kD, lead to the rapid, intraneuronal destruction of the intoxicating BoNT proteases. Here we propose to develop a Clostridium difficile toxin B (TcdB) based vehicle for delivery of the BoNT designer E3-ligases to the cytosol of intoxicated neurons. We propose to test the engineered TcdB agents for reversal of botulism symptoms within cultured neurons and then in mice, likely with a single dose therapy. TcdB has highly evolved features that make it ideal for delivery of BoNT directed designer E3-ligases to the cytosol of botulism intoxicated neurons within patients. TcdB naturally binds to a surface receptor that is broadly expressed in cells, then is internalized by endocytosis and delivers a glucosyltransferase (GT) that inactivates Rho GTPases leading to cell death. This toxin also contains a protease activity that cleaves its enzymatic "cargo" from the "delivery vehicle", and releases it into the cytosol. TcdB has been shown to deliver significant quantities of functional glutathione-S-transferase (GST) to the cell cytosol when the GST is fused to the amino terminus of the toxin GT domain. We have successfully expressed large amounts of full-size, bioactive, recombinant TcdB, and an atoxic variant TcdB, in microbial host cells. In this proposed project, we will develop and test a neuron-specific TcdB by replacing the TcdB receptor binding domain (RBD) with the equivalent RBD domain from BoNT/A. Secondly;we will test whether BoNT/A protease turnover can be accelerated within intoxicated neurons by treatment with a fusion of BoNT/A designer E3-ligase to atoxic TcdB. Finally, an engineered TcdB targeting the BoNT designer ligase to neurons will be tested in the mouse hind limb paresis model for the ability to reverse the symptoms of botulinum toxin induced paralysis. If successful, similar TcdB agents should be readily possible and rapidly available for all botulinum serotypes by simply switching the VHH domain to those having specificity for other BoNT proteases. Secondly, we will have demonstrated that TcdB is a general vector for the delivery of biomolecules to the cytosol of cells- targeted by the specificity of the RBD. Finally, the biomolecular cargo we are delivering to these cells, designer E3-ligases, are simple fusions between a camelid VHH targeting domain and the TrCP F-box, and it is expected that similar agents could be developed to target accelerated turnover of virtually any cytosolic protein for research or therapeutic applications. PUBLIC HEALTH RELEVANCE: Through this proposal, we are directly seeking to develop a single-dose therapeutic cure for botulism, which is caused by several CDC Category A Clostridium botulinum toxins for which no antidote is currently available. If successful, similar agents can be quickly developed for all botulinum serotypes. The same general strategy could be used to deliver therapeutic agents to specific cells to target other pathogenic cytosolic proteins for rapid proteasomal destruction.